The long-term aim of this research is to understand the structural basis underlying the unique substrate specificities of the human CYP2C enzymes. Enzyme inhibition during polytherapy, particularly for CYP2C9, is a general mechanism underlying severe drug-drug interactions with agents such as warfarin and phenytoin. Adverse drug reactions of this type are a major, but potentially avoidable, drain on health care costs in the US. However, our ability to predict inhibitory drug-drug interactions from data obtained in vitro is limited by the lack of high resolution structures and robust pharmacophore models for these P450 enzymes, as well by complications imposed by the observation of atypical Michaelis-Menten kinetics and Type II inhibitor complex formation. This project addresses these limitations with the following specific aims: Specific Aim 1: Construct new CYP2C CoMFA models by determining the inhibition constants for a series of benzbromarone analogs to determine which features confer strong binding interactions with CYP2C9, CYP2C19 and CYP2C8. Specific Aim 2: Determine the effect of pKa and steric factors on strength of type II interactions by measuring binding affinities to CYP2C9 for a series of coumarin-based ligand molecules with and without nitrogen-bearing substituents and with different steric features adjacent to the nitrogen. Specific Aim 3: (a) Determine the structural basis for homotropic cooperative behavior of phenacetin (and pyrene) with human CYP2C9 and CYP2C19 through site-directed mutagenesis, (b) develop complementary NMR approaches to the study of human CYP2C cooperativity. Specific Aim 4: (a) Delineate the role of Arg108 in CYP2C9 substrate selectivity; (b) engineer a soluble, monomeric form of the ultra-stable, iron-nitrogen coordinated Arg108His mutant, and (c) develop histidine scanning mutagenesis and formation of the iron-nitrogen complex as a tool for examining the role of CYP2C9 B-C and F-G loop region mobility in substrate specificity. In this manner, we will bring together organic synthesis, QSAR analysis of Type I and Type II ligands, site-directed mutagenesis, conformational analysis of protein flexibility and, ultimately, crystallography of human CYP2C mutants, to develop an integrated picture of ligand interactions with these important human enzymes that will enable us to assess, in a prospective manner, the potential for inhibitory drug-drug interactions.